This application proposes the simple hypothesis that highly virulent clinical isolates of Mycobacterium tuberculosis, associated as they are with severe inflammation and lung damage, are far more likely as a result to induce regulatory T cell subsets compared to strains of relatively lower virulence. To test this hypothesis we have selected six clinically relevant isolates of an increasing range of virulence. We will use newly developed techniques to follow their emergence, influx, and positioning in the lungs of mice infected by a low dose aerosol with these isolates, use new processes to specifically ablate them, and perform cell transfer studies to directly examine their effects on TH1 immunity. Not only should these studies reveal some basic information regarding the conditions under which regulatory T cells are induced, but they have the potential to provide fundamental information relevant to issues such as the efficacy of new vaccines against clinical strains that acquire high levels of such T cells, and the status of subsequent immunity in individuals infected with highly virulent strains who have successfully been treated with chemotherapy. This then forms the basis of this R21 application. Is the induction of regulatory T cells restricted to this particular strain or is it a more general clinical phenomenon? If just a facet of HN878, is this due to its possession of an unusual cell wall lipid? Is it a specific property of the highly transmitted W-Beijing family? Do such cells directly inhibit effector T cells, or are they designed to reduce inflammation? Efforts are now underway to genomically sequence multiple clinical strains, and we hope to include these strains to be used here in a newly formed sequencing Consortium. As yet, nobody actually knows why certain strains of TB are "virulent" and others less so, and it will require a combination of genomic information compared to basic biological information to try to uncover this. One aspect of virulence could be the ability of the organism to subvert immunity, as we hypothesize here. We aim to determine the kinetics of generation of regulatory T cells in mice infected with a representative panel of clinical isolates of M.tuberculosis, in comparison to effector T cell subsets. We will also track and deplete regulatory T cells in GFP-Foxp3+ and transgenic mice, and further characterize their function in an innovative new adoptive cell transfer system. PUBLIC HEALTH RELEVANCE: Over the past decade the number of isolates of M.tuberculosis that are multi-drug resistant has risen alarmingly. Many of these isolates are from the Beijing/W family which stand out due to the considerable morbidity and mortality they cause worldwide. The basis of this R21 proposal is to characterize if M.tuberculosis strains of extremely high virulence interacted differently with the innate and/or acquired specific host response compared to other strains.